Amino glycoside antibiotics having antiprotozoal and anthelmintic activity

ABSTRACT

In the fermentation production of gentamicin C a plurality of additional antibiotic compounds are obtained. Four of these compounds, gentamicin A, gentamicin B, gentamicin B1 and gentamicin X exhibit significant anti-protozoal and anthelmintic activity while gentamicin B and B1 have significant antibacterial properties with markedly reduced side affects.

This application is a divisional of application Ser. No. 752,375, filedDec. 20, 1976 (now U.S. Pat. No. 4,110,439) which, in turn, is adivisional of application Ser. No. 618,647, filed Oct. 1, 1975 (now U.S.Pat. No. 4,049,796) which in turn is a divisional of application Ser.No. 389,774, filed Aug. 20, 1973 (now U.S. Pat. No. 3,915,955) which inturn is a continuation in-part of application Ser. No. 48,017, filedJune 22, 1970 (now abandoned).

FIELD OF INVENTION

This invention relates to antibiotics which possess antiprotozoal andanthelmintic activity and to methods for their production and use. Moreparticularly, this invention relates to antibiotics co-produced withgentamycin, said antibiotics either in admixture with each other orseparated one from the other possessing antiprotozoal and/oranthelmintic properties.

BACKGROUND OF INVENTION

The preparation, isolation and purification of gentamycin is describedin U.S. Pat. No. 3,091,572. Gentamycin, now known as gentamicin, wasdescribed in the patent as the principal antibiotic component and isreferred to herein as the C-component or gentamicin C. In the patent,reference is made to co-produced antibiotics BA-5 (Fraction A) and BA-3(Fraction B) which in admixture with each other exhibited a low order ofantibacterial activity. These were separated from gentamicin C.

By the utilisation of a particular chromatographic system describedlater herein, we have found that the substances described in U.S. Pat.No. 3,091,572 as co-produced antibiotics BA-3 (Fraction A) and BA-3(Fraction B) are not single entities unto themselves but together theyrepresent a mixture of at least four components. Resolution of thismixture of the co-produced antibiotics into its components (hereinaftercalled gentamicin A, gentamicin B, gentamicin B₁ and gentamicin X), hasled to the discovery that each member of this group possesses surprisingand unexpected applied-use characteristics.

Gentamicin A has been previously isolated in pure form, investigatedchemically and biologically and found to have rather weak antibioticactivity. We have discovered the rather surprising applied-usecharacteristic of gentamicin A, namely, its utility as an antiprotozoalagent. Gentamicin B, gentamicin B₁ and gentamicin X heretofore havenever been isolated one from the other. We have discovered that thesesubstances possess certain valuable properties as antibiotics,anthelmintic and antiprotozoal agents.

BRIEF DESCRIPTION OF THE INVENTION

We have found that the entire mixture of antibiotics produced by thefermentaion of Micromonospora purpurea and Micromonospora echinospora asdescribed in U.S. Pat. No. 3,091,572 may be separated by means of acolumn chromatographic separation process which utilizes the lower layerof a solvent system consisting of methanol, chloroform, and ammoniumhydroxide as the eluting solvent. Prior to chromatography, equal volumesof the solvents are mixed, allowed to settle and the layers separated.Gentamicin C, also known as Garamycin® (Trademark Schering Corporation,Bloomfield, New Jersey) (known to be composed of three components, C₁,C_(1a) and C₂) is first eluted and is followed in turn by gentamicin B₁,gentamicin X, gentamicin B and lastly, gentamicin A. These antibioticsco-produced with gentamicin C and eluted after gentamicin C arestructurally similar to each other as shown in formula I which depictsgross molecular arrangement without regard to spatial considerations.##STR1## wherein R₁, R₂, R₃ and R₄ are as defined above.

Gentamicin A and gentamicin X, and to a lesser extent, gentamicin B andgentamicin B₁, are effective against such protosoa as Entamoebahistolytica, Histomonas meleagridis, Trichomonas foetus and Trichomonasvaginalis. Histomonas meleagridis is the protozoal parasite which causesenterohepatitis, the so-called "blackhead disease" in turkeys. Thisdisease causes serious economic problems in the turkey raising industry.Trichomonas vaginalis is the pathogen most frequently associated withcases of human vaginal infections and is usually a most difficultpathogen to eradicate.

Gentamicin A, gentamicin B, gentamicin B₁ and gentamicin X are alsoeffective as anthelmintic agents against pinworms such as Syphaciaobvelata.

That these substances, gentamicin A, gentamicin B, gentamicin B₁ andgentamicin X, possess valuable antiprotozoal properties is surprising.It has been reported that gentamicin C, which is structurally related tothe aforementioned antibiotics, exhibits only slight activity against T.vaginalis at somewhat high dose ranges. It has been further reported byAlbach et al. in the American Journal of Tropical Medicine and Hygiene,15, 885 (1965) that gentamicin C is relatively ineffective against E.histolytica. All of these gentamicin antibiotics, are members of achemical class of compounds sometimes referred to as aminoglycosides.This class includes such known antibiotics as neomycin, kanamycin,streptomycin and paromomycin. Only paromomycin is known to exhibitantiprotozoal activity to any substantial degree. Furthermore, with theexception of paromomycin, gentamicin A, gentamicin B, gentamicin B₁ andgentamicin X are the only aminoglycosides now known to exhibitanthelmintic activity in reasonably useful dosage ranges.

In view of these properties gentamicin A, gentamicin B, gentamicin B₁and gentamicin X may be used in vitro in wash solution for sanitarypurposes, such as in the cleaning of laboratory equipment contaminatedwith protozoa of the type named above. This is especially true oflaboratory table tops and animal cages and pens from which it is desiredto eradicate susceptible parasitic protozoal and bacterial species. Byvirtue of their in vivo activity, the compounds of this invention may beused in admixture with drinking water or animal feeds to treatlaboratory or domestic animals having intestinal bacterial infections,intestinal protozoal infections, and helmintic infestations, orcombination of the foregoing wherein the infecting organisms aresusceptible to the compounds of this invention. The compounds areespecially useful for intestinal disorders in view of the fact that theyare not appreciably absorbed from the gastro-intestinal tract. Thus,substantial concentrations of therapeutic agent may be ingested withoutcausing toxic manifestations to the host, and at the same time bepresent in quantities sufficient to destroy, or at least tosubstantially inhibit, the infecting organism. For combattinggeneralized (systemic) bacterial infections administration of antibioticis efffected by other than oral means, preferably by injection.

SUMMARY OF THE INVENTION

The invention sought to be patented in one of its process aspectsresides in the concept of resolving a mixture consisting of gentamicinA, gentamicin B, gentamicin B₁ and gentamicin X into the individualantibiotic entities of which the mixture is composed.

The invention sought to be patented in another of its process aspectsresides in the concept of combatting bacterial infections in animalswhich comprises administering to an animal having such an infection atherapeutically effective quantity of a member selected from the groupconsisting of gentamicin B, gentamicin B₁, gentamicin X or theirnon-toxic pharmaceutically acceptable acid addition salts.

The invention sought to be patented in a further process aspect residesin the concept of treating bacterial infections in animals whichcomprises administering to an animal having such an infection atherapeutically effective quantity of a mixture containing as theprincipal active ingredients one or more of the substances of gentamicinB, gentamicin B₁, gentamicin X or their non-toxic pharmaceuticallyacceptable acid addition salt.

The invention sought to be patented in yet another of its processaspects resides in the concept of combatting protozoal infections and/orhelmintic infestations which comprises administering to an animal havingsuch an infection or infestation, a therapeutically effective quantityof a member selected from the group consisting of gentamicin A,gentamicin B, gentamicin B₁, gentamicin X or their non-toxicpharmaceutically acceptable acid addition salts.

The invention sought to be patented in still another of its processaspects resides in the concept of treating protozoal infections and/orhelmintic infestations which comprises administering to an animal,having such an infection or infestation, a therapeutically effectivequantity of a mixture containing as the principal active ingredient oneor more of the substances gentamicin A, gentamicin B, gentamicin B₁ andgentamicin X or the non-toxic pharmaceutically acceptable acid additionsalts thereof.

The invention sought to be patented in one of its composition aspectsresides in the concept of the novel chemical compounds gentamicin B,gentamicin B₁, gentamicin X having the molecular structure ashereinabove described and the non-toxic pharmaceutically acceptablesalts of said compounds.

The invention sought to be patented in another of its compositionaspects resides in the concept of pharmaceutical compositions foreliciting an antibacterial, an anti-protozoal and/or anthelminticresponse which comprises a therapeutically effective quantity a memberselected from the group consisting of gentamicin A, gentamicin B,gentamicin B₁, gentamicin X a non-toxic pharmaceutically acceptable acidaddition salt thereof, or mixtures thereof, and an inert pharmaceuticalcarrier.

DETAILED DESCRIPTION OF THE INVENTION

Gentamicin A, gentamicin B, gentamicin B₁ and gentamicin X have amine orsubstituted amine groups which are capable of forming non-toxic acidaddition salts with organic and inorganic acids. Non-toxic salts aredefined herein as those that do not give rise to detrimental sideeffects when administered at the usual dosage level. Generally, theinorganic acid salts may be formed by adding an amount of dilute (1.0 N)acid to an aqueous solution of antibiotic base to adjust the pH of thesolution to about 4.5 and lyophilizing the resulting solution. Thus, bythe foregoing procedure acid addition salts are formed with hydrochloricacid, sulfuric acid, phosphoric acid and the like including the alkalimetal salts of the dibasic and tribasic acids. In general, the salts ofinorganic acids are water soluble but are substantially insoluble inorganic solvents. However, some such salts (e.g. hydrochlorides) mayhave very limited solubility in polar organic solvents such as loweralcohols. Salts of organic acids, such as, hydrocarbon carboxylic acidsincluding dibasic acids, such as succinic, tartaric, maleic, fumaric,glutaric and the like are usually prepared by reacting an aqueoussolution of the antibiotic with a stoichiometric quantity of acid andeither lyophilizing the resulting solution or precipitating the productwith a miscible organic solvent such as acetone, dioxane,tetrahydrofuran, methanol or the like.

Gentamicin A, gentamicin B, gentamicin B₁ and gentamicin X are obtainedfrom the fermentation as a complex mixture which also includes the majorfermentation product, gentamicin C. The gentamicin "C" componentsconstitute from about 60% to about 80% of the mixture and theco-produced antibiotics constitute the remainder. Thus, in order toobtain the co-produced antibiotics it is advantageous to at leastpartially remove the gentamicin C. There are a substantial number ofresins that are suitable for this purpose. Exemplary of such resins areAmberlite IRA-400, Amberlite IRA-401, Dowex 1-X2, and Dowex 2-X4(Amberlite being a trademark of Rohm and Haas Company, Philadelphia,Pennsylvania) and Dowex being a trademark of Dew Chemical Company,Midland, Michigan). In general, porous, strongly basic quaternary aminetype resins are of utility. Applicants prefer to utilize Dowex 1-X2 inthe hydroxyl cycle.

At the end of the fermentation, the antibiotics produced therein areextracted from the broth which is accomplished by adsorbing the totalantibiotic complex from the fermentation broth onto a cation exchangeresin, preferably of the Amberlite type such as IRC-50. The resin isadvantageously utilized in the ammonium cycle and the crude antibioticmixture eluted with dilute ammonium hydroxide. The eluate is decolorizedby passage through a column containing a decolorizing carbon, or,preferably, by passage through an anion exchange resin column,especially, a resin of the Amberlite IRA type mentioned above. Fordecolorizing the IRC-50 resin eluated IRA-401S resin in the hydroxylcycle is preferred. After decolorization, the eluate is concentrated invacuo to a solution containing from about 30% to about 60% solids andheld for further processing.

At this juncture, the solution contains all of the antibiotics producedin the fermentation and in substantially the ratio produced therein.Removal of the gentamicin C components is effected by adsorbing theconcentrate prepared above on a column of a suitable anion exchangeresin, such as Dowex 1-X2 and selectively desorbing the gentamicin Ccomponents therefrom.

Elution of the gentamicin C components is effected by the use ofdeionized water having from about 50,000 to about 150,000 ohmsresistance. The column may advantageously be connected in series andthrough a conductivity bridge to an automatic fraction collector so thata sharp rise in conductance of the effluent will actuate the fractioncollector. Similarly, a drop in conductance (rise in resistance)followed by a sharp rise in conductance will start the collection of anew fraction. The care with which this column is run, to a substantialdegree, controls the quantity of gentamicin "C", which remains in themixture of co-produced antibiotics and to some degree affects theefficiency of the subsequent (silica gel) column which is used toseparate the co-produced antibiotics one from the other.

The co-produced antibiotics are eluted from the Dowex 1-X2 columnsubsequent to the gentamicin C components and the aqueous eluateconcentrated in vacuo to a suitable volume and lyophilised. At thisstage, it is advantageous to dissolve the antibiotic mixture in hotmethanol and cool while exposing the solution to air. Gentamicin Aadsorbs carbon dioxide from the air forming a sparingly solublecarbonate salt which crystallizes from the methanol solution and isremoved by filtration and dried in vacuo. The filtrate is concentratedto a residue and dissolved in about 3 volumes of the lower phase of asolvent mixture consisting of methanol:chloroform:ammonium hydroxide inthe volume ratio of 1:1:1.

The foregoing solution is placed atop a suitably sized column of silicagel and the column eluted using the above-described solvent mixture anda controlled flow rate. Gentamicin B and gentamicin B₁ may be obtainedin crystalline form by dissolving the residues from the appropriatecolumn fractions in ethanol and concentrating in vacuo to a residue,redissolving the residue in ethanol and re-evaporating the solutionuntil a crystalline suspension is obtained. This procedure yieldssubstantially pure gentamicin B and gentamicin B₁, as ethanol solvateshaving about 1 mole of ethanol per mole of antibiotic. Gentamicin X isusually obtained as an amorphous solid and, in this connection, behaveslike the gentamicin "C" components.

Alternatively, the total antibiotic mixture from the IRA-401S columnwhich includes the gentamicin C components may be subjected to silicagel chromatography. However, the separation effected by this techniqueis not as sharp as that obtained when most of the gentamicin "C" and thegentamicin A are removed prior to silica gel chromatography.

Gentamicin A, gentamicin B, gentamicin B₁, and gentamicin X, after theremoval of the gentamicin C components, may be lyophilized and used as amixture to elicit an antibacterial, antiprotozoal and/or an anthelminticeffect.

The compounds of this invention readily form hydrates and solvates whichare extremely difficult to break. Consequently, the physical constantsfor gentamicin A, gentamicin B and gentamicin B₁ are based uponrelatively stable hydrates or solvates. Further, although gentamicin Band gentamicin B₁ form crystalline ethanol solvates they do not givereproducible melting points but rather tend to soften and eventuallybecome viscous liquids over quite a wide temperature range. Gentamicin Xhas not yet obtained in crystalline form; it is isolated as a whiteamorphous solid which likewise has no discrete melting point.

The antibiotics described herein have physical properties substantiallyas set forth in Table I below:

                                      TABLE I                                     __________________________________________________________________________    Physical Constants                                                                       Elemental                                                                           Analyses      Sulfate Salt                                   Compound   Calculated*                                                                         Found α .sub.D.sup.26                                                                 Analyses                                                                            α .sub.D.sup.26                    __________________________________________________________________________    Gentamicin A                                                                             C = 43.63                                                                           C = 43.60                                                                           +136°  +96°                              Calculated for                                                                           H = 7.93                                                                            H = 7.86                                                                            (C = 1.0,H.sub.2 O)                                                                         (C = 0.3,H.sub.2 O)                      C.sub.18 H.sub.36 N.sub.4 O.sub.10.1.5H.sub.2 O                                          N = 11.31                                                                           N = 11.56                                                               O.sup.1 = 37.13                                                                     O.sup.1 = 37.16                                              Gentamicin B                                                                             C = 47.70                                                                           C = 47.78                                                                           +155°                                                                          C = 30.35                                                                           +116°                             Calculated for                                                                           H = 8.41                                                                            H = 8.38                                                                            (C = 0.3,H.sub.2 O)                                                                   H = 6.59                                                                            (C = 0.3,H.sub.2 O)                      C.sub.19 H.sub.38 N.sub.4 O.sub.10.C.sub.2 H.sub.5 OH                                    N = 10.60                                                                           N = 10.59     N = 7.49                                                  O.sup.1 = 33.29                                                                     O.sup.1 = 33.25                                                                             S = 8.48                                       Gentamicin B.sub.1                                                                       C = 47.22                                                                           C = 48.68                                                                           +161°                                                                          C = 31.26                                                                           +117°                             Calculated for                                                                           H = 8.34                                                                            H = 8.56                                                                            (C = 0.3,H.sub.2 O)                                                                   H = 6.83                                                                            (C = 0.3,H.sub.2 O)                      C.sub.20 H.sub.40 N.sub.4 O.sub.10.C.sub.2 H.sub.5 OH                                    N = 10.41                                                                           N = 10.32     N = 7.34                                                  O.sup.1 = 34.03                                                                     O.sup.1 = 32.44                                                                             S = 8.72                                       Gentamicin X     C = 43.54                                                                           +154°                                                                          C = 31.96                                                                           +121°                             C.sub.19 H.sub.38 N.sub.4 O.sub.10                                                             H = 7.22                                                                            (C = 0.3,H.sub.2 O)                                                                   H = 6.57                                                                            (C = 0.3,H.sub.2 O)                                       N = 9.90      N = 8.64                                                        O.sup.1 = 39.34                                                                             S = 8.70                                       __________________________________________________________________________     .sup.1 All oxygen values are by difference                               

Gentamicin A, gentamicin B, gentamicin B₁ and gentamicin X havecharacteristic infrared absorption spectra in mineral oil (Nujol) asshown by FIG. 1 (gentamicin A), FIG. 3 (gentamicin B), FIG. 5(gentamicin B₁) and FIG. 7 (gentamicin X). These spectra are of therespective antibiotics as sulfate salts.

These antibiotics also have characteristic nuclear magnetic resonancespectra (NMR) as shown by FIG. 2 (gentamicin A), FIG. 4 (gentamicin B),FIG. 6 (gentamicin B₁), and FIG. 8 (gentamicin X). All of the NMRspectra are of the antibiotic free bases; however, the samples ofgentamicin A and gentamicin X were hydrated and those of gentamicin Band gentamicin B₁ are of the ethanol solvate. These spectra wereobtained by the use of a Varian A-60-A spectrometer (Varian Associates,Palo Alto, California) on about 0.4 ml. of a solution (conc. about 20mg/ml) of the antibiotic in deuterium oxide (D₂ O). The spectra arerecorded in parts per million (PPM) from3-(trimethylsilil)-propanesulfonic acid sodium salt, the internalstandard.

In Table II below are set forth the more characteristic peaks from FIGS.1, 3, 5 and 7, respectively.

                  TABLE II                                                        ______________________________________                                        Significant Infrared Absorption Bands                                         ______________________________________                                        Gentamicin A Sulfate                                                          2.9-3.8μ                                                                              (S, V brd) 6.85μ   (Nujol)                                      3.35μ   (Nujol)    7.25μ   (Nujol)                                      3.55μ   (Nujol)    8.50-10.75μ                                                                           (V, S, V brd)                                4.83μ   (W, brd)   12.95μ  (V, W, brd)                                  6.17μ   (M)        13.88μ  (W, brd)                                     6.53μ   (M)                                                                Gentamicin B Sulfate                                                          2.95-3.25μ                                                                            (M-S, brd) 7.26μ   (Nujol)                                      3.40μ   (Nujol)    7.75μ   (W)                                          3.51μ   (Nujol)    8.75-9.75μ                                                                            (S, V brd)                                   4.85μ   (W, brd)   10.28μ  (W-M)                                        6.16μ   (M)        12.95μ  (VW, brd)                                    6.55μ   (M)        13.90μ  (W, brd)                                     6.83μ   (Nujol)                                                            Gentamicin B.sub.1 Sulfate                                                    2.95-3.25μ                                                                            (M-S, brd) 7.25μ   (Nujol)                                      3.38μ   (Nujol)    7.75μ   (W)                                          3.50μ   (Nujol)    8.75-9.75μ                                                                            (S, V brd)                                   4.85μ   (W, brd)   10.27μ  (W-M)                                        6.16μ   (M)        10.75μ  (W)                                          6.55μ   (M)        13.05μ  (V-W, brd)                                   6.83μ   (Nujol)    13.90μ  (W, brd)                                     Gentamicin X Sulfate                                                          3.0-3.3μ                                                                              (S, brd)   7.25μ   (Nujol)                                      3.37-3.51μ                                                                            (Nujol)    7.75μ   (W-M)                                        3.65-4.33μ                                                                            (N, brd)   8.7-10.0μ                                                                             (V-S, brd)                                   4.82μ   (W, brd)   10.30μ  (Brd, shd)                                   5.94μ   (W, shd)   11.50μ  (W, brd)                                     6.17μ   (M)        13.03μ  (V-W, brd)                                   6.54μ   (M)        13.90μ  (Nujol)                                      6.83μ   (Nujol)    15.08μ  (V-W)                                        ______________________________________                                         Notations:                                                                    M = medium,                                                                   S = strong,                                                                   shd. = shoulder,                                                              V = very,                                                                     W = weak,                                                                     brd. = broad.                                                            

ANTIBIOTIC ACTIVITY-IN VITRO

In Table III below is set forth the in vitro minimal inhibitoryconcentration of gentamicin B, gentamicin B₁ and gentamicin X all in thefree base form against a variety of gram positive and gram negativemicroorganisms. The experiments were carried out in a yeast beef brothmedium at pH 7.4.

                  TABLE III                                                       ______________________________________                                        MIC (mcg/ml).sup.1                                                            Organism   Gentamicin B                                                                             Gentamicin B.sub.1                                                                        Gentamicin X                                ______________________________________                                        Aerobacter aero-                                                                          0.08       0.08       --                                          genes                                                                         Bacillus subtilis                                                                        0.8        3.0         0.03                                        Escherichia coli                                                                         0.03-0.3   0.08-0.3    0.3                                         Klebsiella 3.0        3.0         0.75                                        pneumoniae                                                                    Pseudomonas                                                                              0.03-0.08   0.08        0.3-0.75                                   aeruginosa                                                                    Sarcina lutea                                                                            0.3        0.3         --                                          Staphylococcus                                                                           0.3-3.0    0.3-3.0     0.08-0.75                                   aureus                                                                        Streptococcus                                                                            3.0        3.0          0.3-0.75                                   pyogenes                                                                      Shigella sp. M324                                                                        5.0        5.0         --                                          Salmonella sp. 1                                                                         0.8        0.3         0.75                                        Salmonella sp. 8                                                                         0.8        0.3         --                                          Salmonella sp. B                                                                         0.8        0.3         --                                          ______________________________________                                    

ANTIBIOTIC ACTIVITY-IN VIVO

The protective activity of gentamicin B, gentamicin B₁ and gentamicin Xis determined by administering the respective compound subcutaneouslyone-hour after administration of an infecting dose of pathogen and bycounting the survivors 48 hours after infection. Infected non-treatedcontrols die in about 18 to 24 hours. Generally 5-7 dose levels areadministered to mice weighing about 20 grams in groups of 7-10. The PD₅₀values are set forth in Table IV below:

                  TABLE IV                                                        ______________________________________                                        Protective Activity of Gentamicin B, B.sub.1 and X in Mice                    Injecting            PD.sub.50 (mg/kg)                                        Microorganism                                                                           Gentamicin B                                                                             Gentamicin B.sub.1                                                                        Gentamicin X                                 ______________________________________                                        Staphylococcus                                                                aureus Gray                                                                             20.0       30.0        4.0                                          Staphylococcus                                                                aureus W  50.0       50.0        --                                           Staphylococcus                                                                aureus Smith                                                                            5.0        2.5         --                                           Streptococcus                                                                 pyogenes C                                                                              15.0       18.0        --                                           Salmonella                                                                    paratyphi B                                                                             5.0        4.0         --                                           Pseudomonas                                                                   aeruginosa SC                                                                           2.0        2.0         7.0                                          Escherichia                                                                   coli SC   --         --          1.5                                          ______________________________________                                    

ACUTE TOXICITY

The acute toxicity of gentamicin B, gentamicin B₁ and gentamicin X isdetermined in male CF-1 (Carworth Farms) mice weighing about 20 gramseach. The results of this test are set forth in Table V below:

                  TABLE V                                                         ______________________________________                                         Acute Toxicity of Gentamicin B, Gentamicin B.sub.1 and                       Gentamicin X                                                                         LD.sub.50 (mg/kg)                                                      Route    Gentamicin B                                                                              Gentamicin B.sub.1                                                                         Gentamicin X                                ______________________________________                                        I.V.     228         230          225                                         I.P.     1750        1600         >1000                                       S.C.     >1500       >1500                                                    Oral     >250        >250                                                     ______________________________________                                    

IN VITRO ACTIVITY AGAINST TRICHOMONAS VAGINALIS

In Table VI below is set forth the in vitro activity of the compounds ofthis invention against T. vaginalis. The test procedure used wassubstantially as described by R. J. Schnitzer on pages 289-331 inExperimental Chemotherapy, Volume 1, Academic Press, New York (1963).

                  TABLE VI                                                        ______________________________________                                                Minimal 99% Sup-                                                                             Minimal                                                        pression Level (mcg/ml)                                                                     Cidal Level (mcg/ml)                                    Compound  24 hours  48 hours  24 hours                                                                             48 hours                                 ______________________________________                                        Gentamicin A                                                                            10         5-10     25     10                                       Gentamicin B                                                                            25        25        >250   25                                       Gentamicin B.sub.1                                                                      25        10-25     >250   25                                       Gentamicin X                                                                            --        2.5       --     10                                       Garamycin®                                                                          250       100       >250   100                                      ______________________________________                                         STS -- Simplified Tripticase Serum Medium, Baltimore Biological               Laboratories, Baltimore, Maryland.                                       

Against Entamoeba hystolytica, gentamicin X has a minimal inhibitoryconcentration of 4 mcg/ml (48 hours) and a minimal cidal concentrationof 7 mcg/ml (48 hours).

ANTHELMINTIC ACTIVITY IN VIVO

Gentamicin B and gentamicin X exhibit anthelmintic activity againstSyphacia obvelata when tested in groups of seven male Millerton mice bysubstantially the procedures described by O. D. Standen on pages 701-892in Experimental Chemotherapy, Volume 1

                  TABLE VII                                                       ______________________________________                                                                             Mean                                                         % in      %      Worm                                     Compound mg/kg/day  Diet      Infected                                                                             Burden                                   ______________________________________                                        Gentamicin B                                                                           259        0.25      0      0                                        Gentamicin B                                                                           100        0.0625    0      0                                        Gentamicin B                                                                           39         0.02      0      0                                        Gentamicin B                                                                           8          0.008     67     29.5                                     Gentamicin X                                                                           310        0.25      0      0                                        Gentamicin X                                                                           62         0.05      83     27                                       Controls 0          0         100    53                                       Controls 0          0         100    22                                       Controls 0          0         75     6.8                                      ______________________________________                                    

The data set forth in Table VII shows that gentamicin B has anthelminticactivity against Syphacia obvelata at a minimum dosage of 39 mg/kg/daywhen administered to male Millerton mice. Gentamicin X is totallyeffective at 310 mg/kg/day and partially effective at 62 mg/kg/day undersubstantially the same test conditions.

DIMINISHED INCIDENCE OF ATAXIA

Gentamicin B, and to a lesser extent, gentamicin B₁ exhibit a propertythat is quite unusual in amino-glycoside antibiotics. When subjected tothe test procedures described by Weinstein, Wagman and Taber inAntimicrobial Agents and Chemotherapy, 1965, pages 227-231, each ofthese compounds required substantially longer periods of administrationat high doses to cause impairment of the "righting reflex" and theappearance of ataxia. There appears to be a causal relationship betweenhigh doses of amino-glycoside type antibiotics given over an extendedperiod of time and vestibular damage wherein one of the earlymanifestations is ataxia. The test results, in Table VIII, show thatgentamicin B and gentamicin B₁ as compared with gentamicin C (Garamycin)may be used at dosages that are substantially above the normaltherapeutic dose. Moreover, they may be used at such dosages for a timeinterval substantially beyond that normally required to eliminateinfection without causing ataxia; and with minimal, if any, toxicmanifestations. Thus, gentamicin B and gentamicin B₁ represent asubstantial improvement over antibiotics of this class (amino-glycoside)that are currently in use.

                  TABLE VIII                                                      ______________________________________                                        Chronic Toxicity in Cats After Daily Subcutaneous Dosing                                Dose        No. of   Average No. Days                               Antibiotic                                                                              (mg/kg/day) Cats     to become ataxic                               ______________________________________                                        Gentamicin B.sub.1                                                                      240         1        21.0                                                     60          4        69.0                                                     40          4        108.0                                          Garamycin®                                                                          160         8        13.5                                                     40          11       17.5                                                     20          5        31.4                                           Gentamicin B                                                                            240         1        21.0                                                     60          4        81.0                                                     40          4        >312*                                          ______________________________________                                         *Study terminated 2 of the 4 cats had not become ataxic at the end of 385     days, number given is the truncated median.                              

PROPHYLACTIC ACTIVITY OF GENTAMICIN A IN TURKEYS AGAINST HISTOMONASMELEAGRIDIS

Gentamicin A was administered to groups of turkeys at dosage levels of5, 10, and 15 mg. per turkey per day in aqueous solution. Two days afterthe initiation of the drug, the turkeys were inoculated orally with 500Heterakis gallinae ova (the intermediate host carrier of Histomonas). Asingle group of turkeys received 0.035% of gentamicin A in their feedfor six days. Untreated infected controls were also carried in eachexperiment. The test is continued for twelve days and the birds areautopsied on the thirteenth day. Infection is usually manifestedprincipally in two areas, the cecum and the liver which are the areasexamined during the autopsy.

According to the results obtained in the foregoing test, gentamicin Aexhibits prophylactic activity in turkeys against Histomonas meleagridisat a minimum dosage level of 10 mg. per bird, whereas the untreatedcontrols became infected.

EXAMPLE 1 Isolation of Gentamicin C and the Co-produced Antibiotics fromthe Fermentation

Adjust the pH of the fermentation broth obtained as described in U.S.Pat. No. 3,091,572 to pH 2.0 with 6 N sulfuric acid. Stir the mixturefor about 30 minutes and filter through a suitably sized filter whichhas been pre-coated with about a one (1) inch layer of filter aid.Re-adjust the pH of the filtrate to about 7 with 6 N ammonium hydroxideand add oxalic acid to lower the pH to about 4. This quantity of oxalicacid is usually sufficient to precipitate the calcium ions in thefermentation broth as the insoluble calcium oxalate. Adjust the pH toabout 7 with 6 N ammonium hydroxide and stir the mixture for from about1 to about 4 hours at about 20°-35° C. Refilter the mixture using asuitably sized pre-coated filter and save the filtrate. Adsorb theantibiotic mixture on IRC-50 (ammonium cycle) using about 10 grams ofresin per liter of the original broth volume. Wash the resin color freewith deionized water and elute the antibiotics with 2 N ammoniumhydroxide. Concentrate the eluate in vacuo to a solution containingabout 50% solids.

EXAMPLE 2 Separation of Gentamicin C Components from Gentamicin A,Gentamicin B, Gentamicin B₁ and Gentamicin X

Charge 45 cu. ft. of Dowex 1-X2 resin (hydroxyl cycle) to a cleanchromatographic column which is 3 ft. in diameter, at least 8 ft. highand is connected through a conductivity bridge to a fraction collector.Pump deionized water through the resin bed until the effluent has aresistance of at least 65,000 ohms. Charge an aqueous solution of about120 liters containing about 60 kilograms of solids (from Example 1) tothe column and adsorb the antibiotic mixture by permitting theantibiotic solution to pass through the column at the rate of about 2liters per minute.

Develop the column with deionized water at the rate of about 2 litersper minute and collect fractions in accordance with the conductance ofthe eluate. Typically, about seven fractions are collected which varyfrom about 14 to about 750 liters in volume. The first five fractionscomprising about 500 liters contains the bulk of the gentamicin Ccomponents. The remaining two fractions comprising about 1500 to 1600liters contains the bulk of gentamicin A, gentamicin B, gentamicin B₁and gentamicin X. The latter two fractions are combined, concentrated invacuo to a small volume and either lyophilised or spray dried.

In those instances wherein a mixture of the co-produced antibiotics areto be utilized, either the lyophilised or the spray dried product willsuffice.

EXAMPLE 3 Isolation of Gentamicin A

Dissolve 19.8 kilograms of fermentation product from which most of thegentamicin C (Example 2) has been removed in 45 liters of hot methanol.Clarify the solution by filtration. Cool the solution at roomtemperature with exposure to air and seed with gentamicin A. GentamicinA crystallizes as its colorless carbonate salt which is filtered, washedsparingly with methanol and dried at 80° C. in vacuo to obtain 1119grams of the product of this example. The mother liquor and washes arecombined, concentrated to a residue in vacuo and chromatographed asdescribed in Example 4.

EXAMPLE 4 Chromatographic Separation of Gentamicin B Gentamicin B₁ andGentamicin X

Fill two 5'×4" chromatographic columns connected in series with silicagel (e.g. Baker Silica Gel for Chromatography) which has previously beenconditioned by contact with the lower phase of a 1:1:1 mixture ofchloroform, methanol and concentrated ammonium hydroxide. Prepare asolution of 750 grams of the residue from Example 3 in a minimum volume(about 2 liters of the lower layer of the above-mentioned solventmixture). Place the antibiotic solution atop the first column and elutethe column at the rate of about 3 to about 4.5 liters per hour,collecting fractions of approximately two (2) liters. Monitor theeffluent by thin layer chromatography on silica gel plates using thesame solvent system that is being used on the column. Combine thefractions containing like antibiotics and evaporate to a residue. Whenthe amount of gentamicin B eluted tapers off, the column is eluted witha mixture comprising equal volumes of methanol and concentrated ammoniumhydroxide to strip the column. The fractions containing gentamicin B andgentamicin B₁ are dissolved in ethanol and concentrated to a residue.Repetition of the foregoing step will result in a crystalline suspensionof the ethanol solvate of gentamicin B and gentamicin B₁. When theforegoing chromatographic separation is effected, the products areobtained in the amounts and order substantially as shown below:

    ______________________________________                                        Gentamicin C                                                                                      198.6  gms                                                Gentamicin B.sub.1                                                            Gentamicin B.sub.1  42.0   gms    α .sub.D.sup.26°  =                                              157°                                 Gentamicin B.sub.1                                                                                67.8   gms                                                Gentamicin X                                                                  Gentamicin X        12.3   gms    α .sub.D.sup.26°  =                                              153.8°                               Gentamicin X                                                                                      74.9   gms                                                Gentamicin B                                                                  Gentamicin B        85.8   gms    α .sub.D.sup.26°  =                                              132°                                 Gentamicin B                                                                                      165.0  gms                                                Gentamicin A                                                                  ______________________________________                                    

The fractions containing mixtures of antibiotics may berechromatographed to afford more of the individual fractions.

EXAMPLE 3 Preparation of Gentamicin A Sulfate

Dissolve 5.0 grams of gentamicin A in 25 ml. of water and adjust the pHof the solution to 4.5 with 1 N sulfuric acid. Treat the solution withabout 50 milligrams of decolorizing carbon for about 15 minutes andfilter. Pour the filtrate into about 300 milliliters of methanol withvigorous agitation, continue the agitation for about 10-20 minutes andfilter. Wash the precipitate with methanol and dry at about 60° C. invacuo to obtain the product of this Example.

In like manner, the sulfate salt of gentamicin B, gentamicin B₁ andgentamicin X may be prepared. In order to prepare sulfate salts of amixture of gentamicin A, B, B₁ and X, the lyophilised or spray driedproduct of Example 2 is treated as described in this example.

EXAMPLE 6 Preparation of Gentamicin B₁ Hydrochloride

Dissolve 5.0 grams of gentamicin B₁ in 1000 milliters of water. Preparea column containing 500 grams of weakly basic ion exchange resin in thechloride cycle, preferably, IR-45 (Trademarked product of Rohm and Haas,Philadelphia, Penn.). Pass the antibiotic solution through the columnfollowed by an additional 500 milliters of water. Concentrate theeffluent to about 100 milliters in vacuo and lyophilize to obtain theproduct of this example.

In like manner, the hydrochloride salt of gentamicin A, gentamicin B,and gentamicin X may also be prepared.

The tangible embodiments of this invention (i.e. gentamicin A,gentamicin B, gentamicin B₁ and gentamicin X are suitable for oral,topical or parenteral administration. When administered orally they maybe compounded in the form of tablets, capsules, elixirs and the like ormay even be admixed with animal feed. It is in these dosage forms thatthe tangible embodiments of this invention are most effective asanthelminthic and antiprotozoal agents. This is especially true whereinthey are being used to treat intestinal amoebiasis or non-specificdiarrheas. The compounds of this invention may be incorporated into adosage unit as the sole active ingredient or they may be compounded intodosage units in combination with other active ingredients, such asanti-anxiety agents, it being known that infections and infestationsoften cause anxiety reactions in animal species. When the animalsspecies is being treated orally, the tangible embodiments of thisinvention are administered at from about 10 to about 100 mg. perkilogram of body weight per day, preferably divided into from about 2 toabout 4 doses.

For topical administration, the tangible embodiments of this inventionmay be compounded into creams, ointments, suppositories and the like.Such dosage forms should contain from about 1 to about 10% of antibioticand should be administered to the site of the infection from about 2 toabout 4 times a day.

As shown hereinabove, (see Table VIII) gentamicin B, and gentamicin B₁,by virtue of their low ataxic potential, may be used to treatdeep-seated bactorial infections especially those which have proved tobe refractory to other antibacterial agents. It is preferred thatgentamicin B and/or gentamicin B₁ be administered parenterally indosages ranging between about 2 and about 10 mg. per kilogram of bodyweight per day in divided doses.

The examples below set forth some of the various dosage forms into whichthe tangible embodiments of this invention may be compounded.

EXAMPLE 7

    ______________________________________                                        PARENTERAL SOLUTION                                                                             mg/ml      mg/10 ml.                                        ______________________________________                                        Gentamicin B (as the sulfate)                                                                   40 to 200 mg*                                                                            400-2000                                         Methylparaben     1.8 mg.    18 mg.                                           Propylparaben     0.2 mg.     2 mg.                                           Water for Injection q.s.                                                                        1.0 ml.    10.0 ml.                                         ______________________________________                                         *Based on theoretical 100% purity.                                       

Place 70% of the water for injection into a suitable mixing vessel andheat to 70° C. Add the methylparaben and propylparaben and mix untildissolved. Cool the above solution to 25°-30° C. Pass a stream ofnitrogen gas through the solution. Add the gentamicin B (as the sulfate)and mix until dissolved. Bring the solution to final volume. Pass thesolution through a suitable sterilizing filter, employing appropriateaseptic techniques. Fill the solution into suitable sterile containersemploying appropriate aseptic filling techniques.

EXAMPLE 8

    ______________________________________                                        PARENTERAL SOLUTION                                                                             mg/ml      mg/10 ml.                                        ______________________________________                                        Gentamicin A (as the Sulfate)                                                                   40 to 200 mg*                                                                             400-2000                                        Methylparaben     1.80       18.00                                            Propylparaben     0.20       2.00                                             Sodium Bisulfate  3.20       32.0                                             Disodium Edetate, Dihydrate                                                                     0.10       1.00                                             Glacial Acetic Acid                                                                             1.25       12.50                                            Sodium Acetate    2.50       25.00                                            Propylene Glycol  100 to 400 1000-4000                                        Water for Injection q.s.                                                                        1.0 ml     10.00 ml                                         ______________________________________                                         *Based on theoretical 100% purity.                                       

Place 70% of the water for injection into a suitable mixing vessel andheat to 70° C. Add the methylparaben and propylparaben and mix untildissolved. Cool the above solution to 25°-30° C. Pass a stream ofnitrogen gas through the solution. Add the disodium edetate, dihydrate,sodium acetate and glacial ascetic acid; mix thoroughly. Stop thenitrogen sparging, add the sodium bisulfite and mix until dissolved. Addthe propylene glycol and mix thoroughly. Add the gentamicin A (as thesulfate) and mix until dissolved. Bring the solution to final volume.Pass the solution through a suitable sterilizing filter, employingappropriate aseptic techniques. Fill the solution into suitable sterilecontainers employing appropriate aseptic filling techniques.

EXAMPLE 9

    ______________________________________                                        PARENTERAL SOLUTION                                                                             mg/ml      mg/100 ml                                        ______________________________________                                        Gentamicin B.sub.1 (as the Sulfate)                                                             40 to 200 mg*                                                                            4000-20,000                                      Methylparaben     1.80       180                                              Propylparaben     0.20        20                                              Sodium Bisulfite  3.20       320                                              Disodium Edetate, Dihydrate                                                                     0.10        10                                              Glacial Acetic Acid                                                                             1.25       125                                              Sodium Acetate    2.50       250                                              Water for Injection q.s.                                                                          1.00 ml    100 ml                                         ______________________________________                                         *Based on theoretical 100% purity.                                       

Place 70% of the water for injection into a suitable mixing vessel andheat to 70° C. Add the methylparaben and propylparaben and mix untildissolved. Cool the above solution to 25°-30° C. Pass a stream ofnitrogen gas through the solution. Add the disodium edetate, dihydrate,sodium acetate and acetic acid; mix thoroughly. Stop the nitrogen gas,add the sodium bisulfite and mix until dissolved. Add the gentamicin B₁(as the sulfate) and mix until dissolved. Bring the solution to finalvolume. Pass the solution through a suitable sterilizing filter,employing appropriate aseptic techniques. Fill the solution intosuitable sterile containers employing appropriate aseptic fillingtechniques.

EXAMPLE 10

    ______________________________________                                        ORAL SYRUP         PER LITER                                                  ______________________________________                                        Gentamicin A (as the sulfate)                                                                    100        g.*                                             Standard Granulated Sugar                                                                        550                                                        Sorbitol Solution  200                                                        Water, to make     1.0        liter                                           ______________________________________                                         *Based on theoretical 100% purity.                                       

Add the gentamicin A (as the sulfate), granulated sugar and sorbitolsolution, to approximately 350 mls of water contained in a suitablemixing vessel. Mix until a solution is obtained. Add sufficient water tomake 1 liter. Pass the solution through a suitable clarifying filter.

EXAMPLE 11

    ______________________________________                                        ORAL SYRUP         PER LITER                                                  ______________________________________                                        Gentamicin B (as the sulfate)                                                                    100        g.*                                             Granulated sugar   100                                                        Glucose Syrup      44                                                         Sorbitol Solution  400                                                        Glycerin           50                                                         Alcohol            50                                                         Water to make      1.0        liter                                           ______________________________________                                         *Based on theoretical 100% purity.                                       

Add the gentamicin B (as the sulfate), granulated sugar, glucose syrup,sorbitol solution, glycerin, alcohol to approximately 500 ml. of watercontained in a suitable mixing vessel. Mix until a solution is obtained.Add sufficient water to make 1 liter. Pass the solution through asuitable clarifying filter.

EXAMPLE 12

    ______________________________________                                        TOPICAL CREAM           PER KG.                                               ______________________________________                                        Gentamincin A (as the sulfate)                                                                        10 g.-100 g.*                                         Stearic Acid            60 g.                                                 Propylene Glycol Monostearate                                                                         100                                                   Isopropyl Myristate     80                                                    Polyoxyethylene (20) Sorbitan Monopalmitate                                                           60                                                    Sorbitan Solution       20                                                    Water to make           1.0 kg.                                               ______________________________________                                         *Based on theoretical 100% purity.                                       

Add the stearic acid, propylene glycol monostearate, isopropyl myristateand polyoxyethylene (20) sorbitan monopalmitate to a suitable mixingvessel. Heat to 80° C. to melt. Mix.

EXAMPLE 13

    ______________________________________                                        TOPICAL CREAM        PER KG.                                                  ______________________________________                                        Gentamicin B (as the sulfate)                                                                      10 g.-100 g.*                                            Ethoxylated Cetyl/Stearyl Alcohol                                                                  20 g.                                                    Cetyl Alcohol        35                                                       Stearyl Alcohol      35                                                       Petrolatum           200                                                      Mineral Oil          50                                                       Water to make        1.0 kg.                                                  ______________________________________                                         *Based on theoretical 100% purity.                                       

Add the cetyl alcohol, stearyl alcohol, ethoxylated cetyl/stearylalcohol, petrolatum and mineral oil to a suitable mixing vessel. Heat to80° C. to melt. Mix. Add the gentamicin B (as the sulfate) inapproximately 95% of the water heated to 80° C. in a suitable mixingvessel. Mix. Add the melted wax phase to the aqueous phase and mix whilecooling to approximately 40° C. Add sufficient water to make 1 kg. Mixuntil cool.

EXAMPLE 14

    ______________________________________                                        TOPICAL OINTMENT    PER KG.                                                   ______________________________________                                        Gentamicin B.sub.1 (as the sulfate)                                                               10 g.-100 g.*                                             White Petrolatum, to make                                                                         1.0 g.                                                    ______________________________________                                         *Based on theoretical 100% purity.                                       

Melt and heat the petrolatum to 50° C. in a suitable mixing vessel.Remove a portion of the melted petrolatum and make a slurry of thegentamicin B₁ (as the sulfate). Pass the slurry through a suitablecolloid mill and mill until a uniform dispersion is obtained. Add themilled slurry to the remainder of the melted petrolatum and mix untilcool.

EXAMPLE 15

    ______________________________________                                                    TABLETS                                                                           A         B         C                                         Mixture of    10 mg Tab.                                                                              25 mg Tab.                                                                              100 mg Tab.                                 ______________________________________                                        Gentamicin A, B,                                                              B.sub.1 and X (as the Sulfate)                                                               17.50 mg.*                                                                              43.76 mg.*                                                                             175.00 mg.*                                 Lactose, Impalpable                                                           Powder        197.50    171.25    126.00                                      Corn Starch    25.00     25.00     35.00                                      Polyvinylpyrrolidone                                                                         7.50      7.50      10.50                                      Magnesium Stearate                                                                           2.50      2.50      3.50                                                     250.00    250.00    350.00                                      ______________________________________                                         *Based on 60% potency, plus 5% excess.                                   

Prepare a slurry consisting of the mixture of gentamicin A, B, B₁ and X(as the sulfate) from Example 2, lactose and polyvinylpyrrolidone. Spraydry the slurry. Add the corn starch and magnesium stearate, and mix.Compress into tablets using suitable tableting equipment.

EXAMPLE 16

    ______________________________________                                        HARD GELATIN                                                                              A          B          C                                           CAPSULES    10 mg. Cap.                                                                              25 mg. Cap.                                                                              100 mg. Cap.                                ______________________________________                                        Gentamicin A, B,                                                              B.sub.1 and X (as                                                             the sulfate)                                                                              17.50   mg*    43.75 mg.* 175.00                                                                              mg.*                              Lactose, Impalpable                                                           Powder      231.00         205.25     124.25                                  Magnesium Stearate                                                                        1.00           1.00       0.75                                    ______________________________________                                         *Based on 60% potency, plus 5% excess.                                   

Place the mixture of gentamicin A, B, B₁ and X (as the sulfate) andlactose into a suitable mixing bowl and mix. Pass the mixed powdersthrough a mill. Add the mixed milled powders to a suitable mixing vesseland mix again. Pre-mix the magnesium stearate with a portion of thebatch from above. Pass the premixed magnesium stearate to the batch andmix. Fill into empty gelatin capsules using suitable encapsulatingequipment.

EXAMPLE 17

    ______________________________________                                        BOLUS                   100 mg Bolus                                          ______________________________________                                        Mixture of                                                                    Gentamicin A, B, B.sub.1 (as the sulfate)                                                             175.00   mg.*                                         Lactose                 4355.00                                               Corn starch Pregelatinized                                                                            1080.00                                               Magnesium Stearate      90.00                                                 Water                   100.00   ml.                                                                  10,000.00                                             ______________________________________                                         *Based on a 60% potency, plus 5% excess.                                 

Place the mixtures of gentamicin A, B, B₁ and X (as the sulfate),lactose and a portion of the corn starch into a suitable mixing bowl andmix. Prepare a paste of a portion of the corn starch and use this pasteto prepare a damp mass of the above powders. Screen the mass to producegranules. Dry the granules. Reduce the dried granules to specificparticle size. Add the magnesium stearate, mix and compress thegranulation into tablets using suitable tableting equipment.

EXAMPLE 18

    ______________________________________                                        BOLUS                   5 g. Bolus                                            ______________________________________                                        Mixture of                                                                    Gentamicin A, B, B.sub.1 and X (as the sulfate)                                                       8250.00  mg.*                                         Lactose, Impalpable Powder                                                                            8560.00                                               Polyvinylpyrrolidone    900.00                                                Magnesium Stearate      90.00                                                 Corn Starch             200.00                                                                        18,000.00                                                                              mg.                                          ______________________________________                                         *Based on 60% potency, plus 5% excess                                    

Prepare a slurry consisting of the mixture of gentamicin A, B, B₁ and X(as the sulfate), lactose and polyvinylpyrrolidone. Spray dry theslurry. Add the corn starch and magnesium stearate, and mix. Compressinto tablets using suitable tableting equipment.

EXAMPLE 19

    ______________________________________                                        PARENTERAL SOLUTION mg/ml                                                     ______________________________________                                        Gentamicin X (as the sulfate)                                                                     40 to 200  mg.*                                           Methylparaben       1.8        mg.                                            Propylparaben       0.2        mg.                                            Water for Injection q.s.                                                                          1.0        ml.                                            ______________________________________                                         *Based on theoretical 100% purity.                                       

Place 70% of the water for injection into a suitable mixing vessel andheat to 70° C. Add the methylparaben and propylparaben and mix untildissolved. Cool the above solution to 25°-30° C. Pass a stream ofnitrogen gas over the solution. Add the gentamicin X (as the sulfate)and mix until dissolved. Bring the solution to final volume. Pass thesolution through a suitable sterilising filter, employing appropriateaseptic techniques. Fill the solution into suitable sterile containersemploying appropriate aseptic filling techniques.

EXAMPLE 20

    ______________________________________                                        ANIMAL FEED              gm                                                   ______________________________________                                        Mixture of                                                                    Gentamicin A, B, B.sub.1 and X (as the sulfate)                                                         10*                                                 Soybean meal             400                                                  Fish meal                400                                                  Wheat Germ Oil            50                                                  Sorghum Molasses         140                                                  ______________________________________                                         *Based on 100% purity                                                    

Mix all of the above ingredients thoroughly and press into suitablysized pellets, the size of the pellet depending upon animal beingtreated. Mix the pellets with the animal's regular diet in such aquantity that from about 10 to about 100 mg. per kgs. of body weight isconsumed by the animal each day. This composition may be fed tolaboratory animals, such as mice, rats, rabbits, cats, dogs and thelike.

Alternatively, the antibiotic mixture or a single member thereof may bedry mixed with the animal's regular diet so as to provide a therapeuticdose in a normal day's ration. The method is particularly advantageouswhen the antibiotics are used to provide a prophylactic effect such ashas been previously described for turkeys.

EXAMPLE 21

    ______________________________________                                        SUPPOSITORY            5 gm - Suppository                                     ______________________________________                                        Mixture of                                                                    Gentamicin A, B, B.sub.1 and X (as the sulfate)                                                      5        gm.*                                          Water                  10       ml.                                           Gelatin Granule        20       gm.                                           Glycerin               65       gm.                                           ______________________________________                                         *Based on theoretical 100% purity                                        

Dissolve the mixture of gentamicin A, B, B₁ and X (as sulfate) in thewater and add the glycerin with stirring. Finally, add the gelatin andwarm the mixture with stirring until homogeneous. Transfer the mixtureto pre-chilled molds and allow to congeal. This formulation issufficient to prepare twenty 5 gram suppositories.

We claim:
 1. A method of eliciting an anthelmintic response in animalsafflicted with helminthiasis which comprises administering to saidanimals a therapeutically effective quantity for treating helminthiasisof a member selected from the group consisting of gentamicin A,gentamicin B, gentamicin B₁ and gentamicin X, their non-toxic acidaddition salts and mixtures thereof.
 2. A method of claim 1 wherein thetherapeutically effective quantity is from about 10 milligrams to about100 milligrams per kilogram of body weight per day.
 3. A methodaccording to claim 1 wherein gentamicin A is administered.
 4. A methodaccording to claim 1 wherein gentamicin B is administered.
 5. A methodaccording to claim 1 wherein gentamicin B₁ is administered.
 6. A methodaccording to claim 1 wherein gentamicin X is administered.